Rational design of bifunctional protein degrader drugs

泛素(Ub)标签调节细胞内蛋白质的多种性质和功能。蛋白酶体的降解 靶蛋白是Ub蛋白酶体系统(UPS)控制蛋白质含量的一种公认的手段。 被称为Ub E3连接酶的酶通过在ε-E3之间形成异肽键，将Ub与靶蛋白结合。 目的蛋白赖氨酸的氨基和Ub的C-末端甘氨酸的羧基。的共轭 多个瑞银在其七个赖氨酸(K)以及K6、K11、K27、K29、K33、K48和K63中的任何一个形成多Ub链 具有不同作用的UB链存在于所有组织中。最近，几个小组设计了小分子 其一端与E3连接酶结合，另一端与靶蛋白结合，在物理上促进了 目标蛋白质，然后被降解。这种劫持连接酶以泛素化所需蛋白质的行为已经启动 一种名为PROTACs(针对嵌合体的蛋白质分解)的新药。基于PROTAC的方法 治疗药物有几个优点：1)靶标的选择性催化降解；2)弱的转化 结合成选择性PROTAC药物；3)过度表达或突变靶标的降解；以及4)最大 从有限的目标交战中降级。到目前为止，Cereblon和VHL连接酶结合剂是最 通常用作泛素化靶蛋白的载体，如核受体、激酶、转录 因子，以及神经元蛋白tau和α-突触核蛋白。一个主要问题阻碍了新PROTAC的发展 然而，毒品。PROTAC分子的化学优化依赖于合成产物的快速评价 用于指导生产候选药物分子的合成策略的化合物。目前可用的检测方法 是劳动密集型的，不能足够快地向药物化学家提供结果-通常需要一周。 在第一阶段，建立了一种使用Ub连接酶Cereblon和Hdm2的简便体外方法来筛选 潜在的PROTAC药物；PROTAC介导的选定蛋白质的泛素化在体外 在体内模拟观察到的依赖PROTAC的泛素化和这些蛋白质的降解的方式， 实现第一阶段的目标在第二阶段，这种方法的用途将扩大到包括有代表性的 所有Ub连接酶家族的成员(cullin家族、无名指连接酶、Hect家族连接酶和相扑连接酶)， 为PROTAC药物发现增加生化和化学空间。要放大PROTAC屏幕，请使用 将建立以微滴定板为基础的高通量方法来监测体外PROTAC药物发现， 并将使用生化和Ub质谱学蛋白质组学来证明目标蛋白赖氨酸 体外泛素化与体内PROTAC介导的靶蛋白降解相关。 基于微滴定板的PROTAC系统的商业化将对学术研究产生重大影响 以及PROTAC药物发现。

Ubiquitin (Ub) tags regulate multiple properties and functions of proteins in cells. Proteasomal degradation of target proteins is a well-established means whereby the Ub proteasome system (UPS) controls protein content. Enzymes called Ub E3 ligases conjugate Ub to target proteins by forming an isopeptide bond between the ε- amino group of the target protein lysine and the carboxylate of the C-terminal glycine of Ub. Conjugation of multiple Ubs forms poly-Ub chains at any of its seven lysines (K), and K6, K11, K27, K29, K33, K48, and K63 Ub chains having various roles are present in all tissues. Recently, several groups designed small molecules that bind to an E3 ligase at one end and a target protein at the other, physically facilitating ubiquitylation of the target protein, which is then degraded. This hijacking of a ligase to ubiquitylate a desired protein has launched a new class of drug called PROTACs (PROteolysis TArgeting Chimeras). PROTAC-based approaches for therapeutics offer several advantages: 1) selective, catalytic degradation of the target; 2) conversion of weak binders into selective PROTAC drugs; 3) degradation of overexpressed or mutant targets; and 4) maximal degradation from limited target engagement. To date, cereblon and VHL ligase binders have been most commonly used as vehicles to ubiquitylate target proteins such as nuclear receptors, kinases, transcription factors, and neuronal proteins tau and α-synuclein. A major problem has hindered development of new PROTAC drugs, however. Chemical optimization of PROTAC molecules depends on rapid evaluation of synthesized compounds to guide the synthetic strategy for producing drug candidate molecules. Assays currently available are labor intensive and do not provide results to the medicinal chemists fast enough – often, a week is required. In Phase I, a facile in vitro method employing Ub ligases cereblon and HDM2 was developed to screen for potential PROTAC drugs; PROTAC-mediated ubiquitylation of selected proteins was recapitulated in vitro in a way that mimics observed PROTAC-dependent ubiquitination and degradation of these proteins in vivo, achieving the aims of Phase I. In phase II, the utility of this method will be expanded to include representative members of all Ub ligase families (cullin families, RING finger ligases, Hect family ligases, and SUMO ligase), increasing the biochemical and chemical space for PROTAC drug discovery. To scale up PROTAC screens, a microtiter plate-based, high throughput method will be established to monitor in vitro PROTAC drug discovery, and biochemical and Ub mass spec proteomics will be employed to demonstrate that target protein lysines ubiquitylated in vitro are correlated with in vivo PROTAC mediated degradation of target proteins. Commercialization of the microtiter plate based PROTAC system will have a major impact on academic research as well as PROTAC drug discovery.